Part:BBa_K1371015

pT7+birA+accA+pccB+2TM-pT7+sfp+2TM

Substrate supply

There are three enzymes needed in our chassis. Propionyl-CoA carboxylase(pcc), an enzyme needed in the biosynthesis of polyketides, is an enzyme complex containing two components playing the core role in our chassis, of which accA is the α biotinylated subunit functioning as the acetyl-CoA or propionyl-CoA carboxylase while pccB，the β subunit，acts as the carboxyl transferase^[1].

sfp

The sfp gene product, which is part of the surfactin biosynthetic gene cluster in Bacillus subtilis, can effectively modify ACPs from all PKS subclasses as well as related peptidyl carrier protein and aryl carrier protein domains from nonribosomal peptide synthetases (NRPSs). Only with the function of Sfp can propionyl-CoA be linked to ACPs, which is the basement of the reactions.

BirA

BirA is a biotin ligase that improves the activity of accA, acts as an enhancer like sfp, the phosphopantetheinyl transferase facilitating posttranslational modification of the DEBS proteins in E.coli BL21(DE3) ^[2]. With the pcc and sfp genes transformed into E.coli BL21(DE3), it will express DEBS genes at the comparable level to S. erythraea or S.coelicolor ^[2].

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 2946
  Illegal BglII site found at 3362
  Illegal BamHI site found at 1489
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1880
  Illegal NgoMIV site found at 1895
  Illegal NgoMIV site found at 3610
  Illegal NgoMIV site found at 3687
  Illegal AgeI site found at 2252
  Illegal AgeI site found at 2384
  Illegal AgeI site found at 2579
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 1712
  Illegal BsaI.rc site found at 2426
  Illegal BsaI.rc site found at 3826
  Illegal SapI.rc site found at 2039
  

Results

1. Construction

Figure3: Analysis of the chassis we constructed. The 8.5kb plasmid pSB4A5 was digest by EcoRⅠand PstⅠinto two pieces, the 3.4kb backbone and the 5.2kb desired gene. M: 1kb marker; lane1、2 are parallel sample.

Figure 4:Analysis of the two devices transformed into E.coli BL21(DE3), the 15kb device 1 pSB1C3 and the 8.5kb device 2 pSB4A5. M1: 1kb marker; M2 DL15,000 marker; lane1~8: sample1~8 of E.coli BL21(DE3) transformed with both devices.

2. Transcription level

To test the transcription level of our desired genes, we did a series of RT PCR. Besides, as birA is an endogenous gene of E.coli BL21(DE3), we didn’t test it. From figure 5 we could draw a conclusion that accA, pccB and sfp were transcripted as expected.

Figure 5: Transcription level of accA、pccB and sfp, with 16S rRNA2 as the reference gene. ACC: accA; PCC: pccB; SFP: sfp.

3. SDS-PAGE

We detected target protein of the metabolically engineered strain of E. coli BL21(DE3) by SDS-PAGE.

Figure 6: Analysis of the cell disruption product, from which we can see accA、birA and sfp were produced. PccB was not found, but the result of mass spectrometry analysis shows that the expected product triketide lactone was produced, meaning that pccB was translated successfully in our engineered E.coli, too.

Conclusion

We constructed the chassis during August and detected whether it works as expected in the early September. From result 1 and result 2, we can safely draw a conclusion that our chassis was successfully constructed. The later MS analysis shows that it works as expected. As the first desired product triketide lactone was detected and proved our chassis does work, we will see whether it works with other polyketide synthase we transformed into E.coli BL21(DE3).